SAMHSA Releases New Working Definition of 'Recovery'

Deborah Brauser

December 28, 2011 — After considering more than 1,400 comments during a public feedback period this summer, the Substance Abuse and Mental Health Services Administration (SAMHSA) has released a new working definition of "recovery" from mental disorders and substance use disorders.

The organization began consulting members of the behavioral healthcare community more than 1 year ago to develop both major guiding principles and a definition that would capture "the essential, common experiences of those recovering," they write.

The new definition, which was published on SAMHSA's Web site on December 22, now states that recovery is a "process of change through which individuals improve their health and wellness, live a self-directed life, and strive to reach their full potential."

"Over the years it has become increasingly apparent that a practical, comprehensive working definition of recovery would enable policy makers, providers, and others to better design, deliver, and measure integrated and holistic services to those in need," Pamela S. Hyde, SAMHSA administrator, said in a news release.

"I believe SAMHSA has achieved a significant milestone in promoting greater public awareness and appreciation for the importance of recovery, and widespread support for the services that can make it a reality for millions of Americans," added Ms. Hyde.

Public Feedback

In August 2010, SAMHSA invited "mental health consumers and individuals in addiction recovery" to a meeting to discuss the need for a new definition of recovery as part of its Recovery Support Strategic Initiative. There, the participants developed the first draft of a definition as well as a draft of guiding principles of recovery.

Over the following 12 months, SAMHSA worked with other participants at meetings and conferences to clarify and revise the drafts. The first official working definition and principles were then posted on a Web site blog in August 2011, along with an invitation for public feedback.

The initial blog post received 287 comments, with more than 1,200 additional comments left on SAMHSA's Feedback Forums.

Altogether, 1,000 participants left comments; the participants included representatives from organizations such as the National Alliance on Mental Illness (NAMI) and the National Association of State Alcohol and Drug Abuse Directors (NASADAD).

NAMI and NASADAD Respond

"SAMHSA's 10 'Guiding Principles of Recovery' are laudable as a vision to aspire to," Michael J. Fitzpatrick, executive director of NAMI, wrote at that time in one of the blog's comments. "Unfortunately, these principles fail to acknowledge that some people living with the most serious mental illnesses are sometimes limited or unable to exercise self-determination."

He added that neglect or lack of treatment can lead to "horrendous consequences," including large numbers of those with mental illnesses being incarcerated in jails and prisons, "boarded" in emergency departments, or becoming homeless.

"The solution to these problems is a system that is individualized, respectful, allows people to exercise personal choice and autonomy whenever possible, but also has the capacity to intervene and provide humane, compassionate assistance to people when they most need it," wrote Mr. Fitzpatrick.

He also suggested that the important role families play in helping adults living with mental illness should be acknowledged and that the guidelines should emphasize that recovery means different things for different people.

Robert I. L. Morrison, executive director of NASADAD, wrote in his comments that more work should be done in understanding current state-funded recovery services and that SAMHSA should draw from states' experiences with the various Access to Recovery models already implemented.

"In addition, we agree there are commonalities between recovery from mental and substance use disorders that are important to understand, particularly in the context of reimbursement strategies," wrote Mr. Morrison.

He added that it should also be acknowledged that recovery experiences may be different for these people.

SAMSHA noted that many of these comments have been incorporated into the current working definition and principles.

"The response to our request for feedback was tremendous and clearly demonstrated the field's interest and concern on this important issue. Over 8,500 votes were also cast in support of the ideas on the forums," they write.

New Guidelines

The new guiding principles now state that recovery:

• emerges from hope;
• is person-driven;
• occurs through many pathways;
• is holistic;
• is supported by peers and allies;
• is supported through relationships and social networks;
• is culturally based;
• is supported by addressing trauma;
• involves the individual, family, and community; and
• is based on respect.

Along with the definition and principles, SAMHSA has also created "4 major dimensions that support a life in recovery," consisting of health, home, purpose, and community.

Further, they added a preamble emphasizing that "there are many different pathways to recovery, and each individual determines his or her own way."

SAMHSA notes that although several effective models exist for promoting recovery, more work is needed to make sure services and systems are put into place in every state.

"Drawing on research, practice, and personal experience of recovering individuals, within the context of health reform, SAMHSA will lead efforts to advance the understanding of recovery and ensure that vital recovery supports and services are available and accessible to all who need and want them."

The full report was published online December 22 on SAMHSA's Web site.

Source

Natural Variation in Drug Susceptibility to HCV Polymerase Inhibitors in Treatment-naïve HCV Patient Isolates

From Journal of Viral Hepatitis

S.-C. C. Sun; A. Bae; X. Qi; J. Harris; K. A. Wong; M. D. Miller; H. Mo

Posted: 12/27/2011; J Viral Hepat. 2011;18(12):861-870. © 2011 Blackwell Publishing

Abstract and Introduction

Abstract

To assess the natural variation in drug susceptibility among treatment-naïve hepatitis C virus (HCV) patient isolates, the susceptibilities of chimeric replicons carrying the HCV NS5B polymerase from up to 51 patient isolates against a panel of diverse HCV nonnucleoside polymerase inhibitors were evaluated using a replicon-based transient replication assay. Some patient to patient variation in susceptibility to the panel of three HCV nonnucleoside polymerase inhibitors was observed. Linear regression and correlation analyses revealed no correlations among the susceptibilities to the polymerase inhibitors tested. Our results suggest that variable antiviral responses to HCV nonnucleoside polymerase inhibitors may be observed because of the natural variation in baseline susceptibility. In addition, the lack of correlation among the susceptibilities to three classes of HCV polymerase inhibitors evaluated here supports their possible combined use in a combination therapy strategy

Introduction

Hepatitis C virus (HCV) is a positive-strand RNA virus and is estimated to infect over 170 million people worldwide.^[1,2] To date pegylated interferon-α plus ribavirin, the current standard of therapy for HCV infection, is associated with incomplete efficacy and various side effects.^[3–6] Therefore, intense effort and time have been devoted to the discovery and development of novel and selective small-molecule inhibitors of HCV. Although no direct small-molecule antivirals have yet been approved for therapeutic use, the NS3 protease and NS5B polymerase are considered to be prime targets, and inhibitors of each enzyme have shown strong antiviral activity in early clinical trials.^[7–11]

HCV RNA-dependent RNA polymerase is the essential enzyme for replication of HCV RNA. A number of HCV polymerase inhibitors have been discovered; some have advanced to phase I/II clinical trials and have demonstrated antiviral activity in HCV-infected subjects in monotherapy.^[9,10,12] Among these HCV polymerase inhibitors, a number of nucleoside analogues (2'-Me-C, R1479, NM283, R1626 and R7128) bind to the active site of the HCV polymerase.^[13–15] In vitro resistance selection in the presence of nucleoside analogues identified a S282T resistance mutation for 2'-Me-C, and two (S96T and N142T) for R1479.^[16] The S282T mutation has also been reported in virologic breakthrough in patients on an NM283 (Idenix Pharmaceuticals, Cambridge, MA, USA) clinical trial.^[17,18] In addition to the active site, there are four allosteric binding sites for nonnucleoside inhibitors (NNIs) within the NS5B polymerase including: palm I (palm domain near the active site), palm II (partially overlapping palm I and towards the active site), thumb I (thumb domain near the fingertips) and thumb II (the outer surface of the thumb domain).^[19–21] First, the benzothiadiazine and acylpyrrolidine class of NNIs bind to the palm I domain of the polymerase.^[7,20] Resistance selection in the presence of benzothiadiazines revealed amino acid substitutions at residues 411, 414, 448, 451, 553, 554, 555, 556 and 559.^[22–25] Second, the benzofuran class of NNIs (HCV-796; Wyeth/Viropharma) binds to the palm II domain. HCV-796 (benzofuran) has been reported to select a major resistance mutation at amino acid residue 316 in an in vitro replicon system, and in early clinical trials.^[26,27] Distinct from the above HCV polymerase inhibitors, benzimidazoles and indoles bind to the thumb I site.^[28] Reduced susceptibility to this class of compounds was associated with the selection of mutations at residues 495 and 496 of the NS5B polymerase.^[28] Lastly, the thiophene carboxylic acids bind to the thumb II site.^[20,29,30] The most frequent mutations observed in vitro and in vivo for this class are located at amino acid position 423 (M423T/V/I) followed by positions 419 (L419M) and 482 (I482L).^[20,30,31]

HCV is characterized by a high degree of genetic diversity.^[32,33] The nucleotide sequences among the six different genotypes differ at 30–35% of the nucleotide sites.^[34,35] Each of the six major genotypes of HCV contains a series of more closely related subtypes that typically differ from each other by 20–25% in nucleotide sequences. Furthermore, 5–8% sequence divergence was observed between individual strains (variants) of HCV within a given subtype (GT-1a and GT-1b). It is possible that this high degree of natural sequence variation may have an impact on the susceptibility of patient isolates to HCV NS5B inhibitors, possibly, influencing the clinical response to direct antivirals. Therefore, it is of interest to test drug candidates against circulating genotypes from a variety of clinical isolates. In the present study, the susceptibilities of a panel of patient-derived NS5B polymerase to a number of nonnucleoside polymerase inhibitors were evaluated using a transient replication assay. The correlations in drug susceptibility among the different polymerase inhibitors were analysed.

Materials and Methods

Clinical Isolates

Patient serum samples were obtained from untreated GT-1a and GT-1b HCV-infected patients. All patients gave informed consent and originated from the USA.

Compounds

The NNIs benzofuran (NNI-1), benzothiadiazine (NNI-2) and thiophene carboxylic acid (NNI-3) were synthesized at Gilead Sciences, Inc. (Foster City, CA, USA).

Construction of NS5B Shuttle Vector

The genotype 1b-Con-1 subgenomic replicon construct 1b-PI-luc used to create the shuttle vectors has been described by Friebe et al.^[36] The components of the replicon are depicted in Fig. 1. A poliovirus IRES element was added at the 5' end after the HCV 5'NTR to increase firefly luciferase translation and RNA replication. Translation of HCV replicon from NS3 to NS5B is driven by the EMCV IRES. Three adaptive mutations, two in NS3 (E1202G+T1280I) and one in NS4B (K1846T) were introduced for efficient replication.^[37] Luciferase activity was used as an end-point readout.

Figure 1. Design of shuttle vector. Vector used for GT-1a and GT-1b NS5B gene insertion from the clinical isolates. The backbone of the shuttle vector is from 1b-Con-1.

To clone NS5B from patient isolates, a BclI site located at the 9th amino acid residue of NS5B was used as the 5'-end cloning site (Fig. 1). An AseI site was inserted directly after the TGA stop codon of NS5B, giving an insertion of ATTAAT at the 5' end of the 3' NTR. The restriction site was generated by site-directed mutagenesis using a QuickChange XL mutagenesis kit (Agilent, Santa Clara, CA, USA). The 1.0-kb fragment between two BglII sites in NS5B was removed to prevent contamination of recombinant shuttle vectors with the parental NS5B gene in luciferase readouts (Fig. 1).

NS5B Gene Isolations and Subcloning to Shuttle Vector

Viral RNA was isolated from 140 μL of serum from HCV-infected subjects using the QiaAmp Viral RNA isolation kit (Qiagen, Valencia, CA, USA) according to the supplier's instructions. cDNA was synthesized in a 20-μL reaction containing 0.2 mm of each dNTP, 0.125 μm of reverse primers and 50 U/μL MonsterScript reverse transcriptase (Epicentre, Madison, WI, USA) as recommended by the manufacturer. Ten microlitres of the RNA isolated from 140 μL of serum was denatured at 65 °C for 1 min, chilled on ice and added as template for reverse transcription. The reverse transcription reaction was incubated at 50 °C (1a) or 54 °C (1b) for 10 min and then at 60 °C for 40 min. Subsequently, the reverse transcriptase was heat inactivated at 90 °C for 2 min. The cDNA was used as template for PCR amplification of the NS5B gene.

A nested PCR strategy was used with genotype-specific primers to amplify the NS5B gene that was then used as a template for sequencing. GT-1a was amplified using primers 1aNS5B-5'7380-5' GAA TCA ACC CTA TCT ACT GCC TTG GCC GAG C-3' and 1aNS5B-3'9386-5' CTA AGA GGC CGG AGT GTT TAC-3' for the first round of PCR, and primers 1aNS5B-5'-7419-5'- TTT GGC AGC TCC TCA ACT TCC GG-3' and 1aNS5B-3'-9372-5' GAG TGT TTA CCC CAA CCT TCA TCG-3' for nested PCR. GT-1b was amplified using primers 1bNS5B-5'7595 -5' TCG TAC TCC TCC ATG CCC CCC CTT GA-3' and 1bNS5B-3'9459 -5' CCT ATT GGC CTG GAG TGT TTA GCT C-3' for the first round of PCR, and primers 1bNS5B-5'-7637-5'- GAT CTC AGC GAC GGG TCT TGG TC-3' and 1bNS5B-3'-9424-5' TTG GGG AGC AGG TAG ATG CCT AC-3' for nested PCR. PCR parameters were as follows for both genotypes: 94 °C, 2 min, 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2:10 min for first round PCR and 94 °C, 2 min, 35 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 2:10 min for nested PCR. All nested PCRs were performed using the High Fidelity Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). PCR products were purified on a QiaQuick PCR clean-up column (Qiagen), according to the supplier's recommendations. The cycle sequencing of purified PCR product was conducted by ABI BigDye Terminator Sequencing technology (Applied Biosystems, Inc. Carlsbad, CA, USA) with 50 ng of purified PCR product. DNA sequencing conditions were 96 °C for 1 min, followed by 25 cycles of 96 °C for 10 s, 50 °C for 5 s and 60 °C for 4 min. The dried products were then denatured in Hi-Di formamide and run on an ABI 3100 genetic analyzer. DNA sequences were analysed with the Sequencher 4.0 program. Reference sequences of 1a-H77 and 1b-Con-1 were used as comparators to report any changes in the clinical isolates for GT-1a and GT-1b, respectively. Amino acid (AA) sequence analyses were performed from AA 1-591 and AA 1-582 of the NS5B gene for GT-1a and GT-1b, respectively.

The final PCR products for cloning into the NS5B shuttle vector were amplified from the above PCR products with primers containing 5' BclI and 3' AseI restriction sites. The final PCR was performed in the buffer supplied with the polymerase, plus 1.25 mm MgCl₂, 0.3 mm each primer. After digestion with AseI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 3 h and subsequent BclI digestion at 50 °C for 3 h, the PCR product was cleaned with a QiaQuick PCR column (Qiagen) according to the supplier's recommendations. The shuttle vector DNA was similarly digested and gel purified with QIAEX II Gel Extraction kit (Qiagen) according to manufacturer's protocol. Purified shuttle vector DNA was then treated with shrimp alkaline phosphatase (Roche) for 15 min at 37 °C and then 65 °C for 15 min to heat inactivate the enzyme. Ligations were performed with 100 ng each of vector and insert DNA in 20 μL of 50 mM Tris–Cl, pH 7.8, 10 m MgCl2, 10 mM DTT, 1 mm ATP, 25 μg/mL bovine serum albumin and 0.15 Weiss units of T4 DNA ligase (New England Biolabs) at 16 °C, overnight.

Plasmid Purification and RNA Synthesis

The ligation reaction was coprecipitated with Pellet Paint® Co-Precipitant (Novagen, San Diego, CA, USA) according to the supplier's recommendations. The precipitated DNA was then resuspended in 2 μL of water and transfected by electroporation into Escherichia coli ElectroTen-Blue® Electroporation Competent Cells (Agilent) according to the supplier's recommendations. Ten per cent of the transformation mixture was plated on antibiotic selection plates to determine the transformation efficiency and the remaining transformants were expanded in liquid culture to propagate the quasi-species pool. Plasmid DNA from each overnight culture was then purified using two QiaPrep mini spin columns (Qiagen). Plasmid DNA was linearized by digestion with ScaI, and purified by extraction once with an equal volume of phenol/chloroform (1/1). DNA was precipitated with ethanol, and template RNA was synthesized using a T7 Megascript RNA synthesis kit (Ambion, Austin, TX, USA) according to the supplier's instructions. After lithium chloride precipitation, RNA was used for the transient replication assays.

Transient Replication Assay

The Huh-7 cells used in the transient replication assay were derived from a cured replicon cell clone as described previously,^[36] referred to as Lunet cells. For all experiments, Lunet cells were grown to a density of (6–10) × 10⁴ cells/cm² in Dulbecco's minimal essential medium (DMEM; Invitrogen) containing 10% foetal bovine serum (HyClone, Waltham, MA, USA), penicillin (100 U/mL)/streptomycin (100 μg/mL) (Gibco/Invitrogen) and 100 μM MEM Non-Essential Amino Acids Solution (Invitrogen). Cells were harvested by trypsinization and washed twice with ice-cold PBS; cell concentration was adjusted to 1 × 10⁷ cells/mL with ice-cold PBS, and 0.4 mL was transferred to a cold cuvette with a 0.4-cm gap, along with 5–10 μg of template RNA. Electroporation was performed with a Gene Pulser II (BioRad, Hercules, CA, USA) at 960 μF and 270 V using 1 manual pulse. Transfected cells were diluted to 2 × 10⁵ cells/mL and plated in 96-well plates at 2 × 10⁴ cells per well in complete DMEM medium. A duplicate plate was analysed for luciferase activity at 4 h posttransfection for transfection efficiency normalization. Compounds (or DMSO alone as a control) were added 24 h posttransfection in threefold dilutions at a final DMSO concentration of 0.5% (v/v). Firefly luciferase reporter signal was read 72 h after addition of compounds using the Luciferase assay system (Promega, Madison, WI, USA) with a Victor Luminometer (Perkin-Elmer, Waltham, MA, USA).

EC₅₀ Determination and Replication Capacity of HCV NS5B Clinical Isolates

The EC₅₀ values were assessed as the compound concentration at which a 50% reduction in the level of firefly reporter activity was observed when compared with control samples in the absence of compound. Dose response curves and EC₅₀ values were generated by using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA) with nonlinear regression analysis. The signal-to-noise window was determined as the ratio of luciferase activity from cells treated with 0.5% DMSO vs activity from cells treated with 500 nM of BILN-2061 in 0.5% DMSO.

The replication level of either reference strains (1b-Con-1) or chimera replicons derived transiently from clinical isolates was determined as the ratio of the firefly luciferase signal at day 4 to that at 4 h postelectroporation, to normalize for transfection efficiency. The replication capacity of each chimera replicon derived from clinical isolates was expressed as their normalized replication efficiency when compared with that of the reference strain (1b-Con-1) within the same experiment.

Statistical Regression Analysis of Correlation Between Different HCV Inhibitors

The mean EC₅₀ values derived from each individual treatment-naïve HCV patient were compared in a statistical analysis to determine the correlation between different HCV inhibitors. This was calculated using the Pearson product-moment correlation coefficient (r) and the coefficient of determination (r²) of linear regression analysis. Both the correlation and linear regression analyses were performed using GraphPad Prism 4.0 (GraphPad Software).

Results

Characterization of the Phenotypic Assays

The plasmid used for the construction of the NS5B designated as 1b-PI-luc, includes three adaptive mutations (E1202G, T1280I and K1847T) and a poliovirus IRES element that has been shown to enhance both translation of the luciferase gene and RNA replication.^[36] This replicon replicated to high levels in Lunet cells. The NS5B shuttle vector was modified by addition of the 3' restriction site (AseI) for insertion of NS5B genes from clinical isolates. Insertion of the new restriction site did not adversely affect the replication capacity of the replicon (data not shown).

To evaluate the reproducibility of the NS5B phenotypic assay, the susceptibility of reference replicon (1b-Con-1) to the thiophene carboxylic acid, benzofuran and benzothiadiazine NNIs was obtained from between 15 and 16 independent determinants. Table 1 summarizes the mean EC₅₀ values, standard deviations, the 95% confidence intervals, and the maximum fold changes for the NNIs. The assay was highly reproducible with the EC₅₀ determinations from independent assays differing by <2-fold with respect to the mean EC₅₀ for each drug.

The Replication of the Chimeric Replicons Carrying Heterologous NS5B From Clinical Isolates

To test the clinical isolates, the HCV NS5B polymerase gene was amplified from 55 clinical isolates from untreated patients infected with HCV (41 GT-1a and 14 GT-1b) and were ligated into the NS5B shuttle vector. By mimicking the intrinsic HCV heterogeneity present in patients, these pooled populations of molecules were transfected into Lunet cells. The replication capacity of the patient isolates was determined by comparing the luciferase activity in the test sample to the parental 1b-PI-Luc at day 4 in a transient replication assay. As shown in Fig. 2, the replication capacity of the chimeric replicons harboring the NS5B genes from patient sera ranged from 0.01% to 50% of the wild-type 1b-Con-1. In this assay, the luciferase signal-to-noise ratio of the parental 1b-PI-Luc was high (>1000, data not shown). EC₅₀ values can be accurately determined if the clinical sample can replicate at 0.01% of the wild-type replicon (corresponding to signal/noise ratio of >10). Among the 55 patient isolates, 51 of 55 (92.7%) demonstrated sufficient replication levels to allow for drug susceptibility determination while the remaining four clinical isolates for NS5B phenotypic analysis either did not replicate or replicated at a level too low to allow for accurate EC₅₀ determination.

Figure 2. Relative replication capacity of chimeric NS5B clinical isolates from 52 HCV treatment-naïve patients.

Susceptibility of Treatment-naïve Clinical Isolates to HCV Polymerase Inhibitors

To investigate the natural variation in drug susceptibility of the baseline clinical isolates, the chimeric replicons were tested for their susceptibilities to three HCV NNIs. Susceptibilities to the thumb II inhibitor thiophene carboxylic acid were comparable to the laboratory strain 1b-Con-1 (Fig. 3a and Table 1). Thirty-nine of these samples were GT-1a and eight were GT-1b. Four of the clinical isolates failed to generate an EC₅₀ value because of poor curve fitting. The EC₅₀ values ranged from 100 to 764 nM with a mean EC₅₀ of 322 ± 148 nM for GT-1a and 344 ± 148 nM for GT-1b patients. Thus, no difference in susceptibility to the thiophene carboxylic acid tested was observed between GT-1a and GT-1b.

Against the benzofuran, the majority of the GT-1 isolates (45 of 51) were slightly more sensitive to the benzofuran than the laboratory stain 1b-Con-1 (Fig. 3b and Table 1). The EC₅₀ ranged from 1.3 to 13 nM and the mean EC₅₀ values were 5.9 ± 2.9 nM from 39 GT-1a patients and 5.8 ± 1.7 nM from 12 GT-1b patients, which were slightly lower than that of the laboratory strain 1b-Con-1 (9.74 nM. Similar to the thiophene carboxylic acid, no significant difference in sensitivity between GT-1a and GT-1b was observed for the benzofuran.

Figure 3. Susceptibility to HCV polymerase inhibitors of treatment-naïve HCV NS5B clinical isolates indicates as EC₅₀ values. (a) A total of 47 NS5B clinical isolates (39 GT-1a and 8 GT-1b) were tested to determine the sensitivity to thiophene. The mean EC₅₀ is 322 ± 148 nM from GT-1a patients and 344 ± 148 nM from GT-1b patients. (b) A total of 51 NS5B clinical isolates (39 GT-1a and 12 GT-1b) were tested to determine the sensitivity to benzofuran. The mean EC₅₀ is 5.9 ± 2.9 nM from GT-1a patients and 5.8 ± 1.7 nM from GT-1b patients. (c) A total of 44 NS5B clinical isolates (33 GT-1a and 11 GT-1b) were tested to determine the sensitivity to benzothiadiazine. The mean EC₅₀ is 302 ± 165 nM from GT-1a patients and 130 ± 44 nM from GT-1b patients.

In contrast, the GT-1a clinical isolates showed a decreased sensitivity to the palm I inhibitor benzothiadiazine compared to GT-1b clinical isolates, and the laboratory strain 1b-Con-1 (Fig. 3c and Table 1). The reduced susceptibility to the compound ranged from 1.1- to 3.4-fold in EC₅₀ values when compared to the mean of GT-1b clinical isolates. We were unable to obtain reportable EC₅₀ values for seven of the clinical isolates because of poor curve fitting. The mean EC₅₀ was 302 ± 165 nM from 33 GT-1a and 130 ± 44 nM from 11 GT-1b patients.

As shown in Fig. 3 and Table 2, some natural variation in susceptibility among these isolates was observed with all three different classes of HCV polymerase inhibitors tested (benzofuran, benzothiadiazine and thiophene carboxylic acid). The EC₅₀ values in the 95th percentile were 4.5-, 6- and 7.5-fold higher than the 5th percentile EC₅₀ values for the thiophene carboxylic acid, benzofuran, and benzothiadiazine compounds, respectively.

Genetic Diversity of Variants Within Clinical Isolates

Population sequence analysis of full-length NS5B was performed for all 55 baseline clinical isolates, and each clinical isolate was aligned against respective subtype reference, GT-1a: 1a H77-AF009606 or GT-1b: 1b-Con-1-AJ238799, to identify differences between patient and reference amino acid sequences. Genotypic analysis revealed intrinsic genetic diversity among the clinical isolates, but did not identify any known resistance mutations that have been reported to be associated with resistance to any of the three classes of compounds tested in 52 of 55 isolates including all 41 GT-1a isolates (Table 3). In contrast, 1 of 14 GT-1b isolates harboured S556S/G as a single mutant/wild-type mixture; S556S/G is known to confer resistance to the benzothiadiazines (Table 3). This isolate had EC₅₀ values of 546.5, 5.42 and 144.6 nM for the thiophene carboxylic acid, benzofuran and benzothiadiazine, respectively (Table 4). The EC₅₀ value against the benzothiadiazine was the third highest among the 11 GT-1b isolates, but was within the natural variation. In addition, 2 of 14 (14%) GT-1b isolates contained both C316N and S556G mutants, known to confer low-level of resistance to the benzofuran and/or benzothiadiazine classes (Table 3). The drug susceptibility data were available from only one of these two isolates because the chimeric replicons from the second replicated poorly. The EC₅₀ values were 9.14 nM for the benzofuran, the highest among the 12 GT-1b isolates tested, and 112.27 nM for the benzothiadiazine, comparable to other GT-1b isolates (Table 4).

Linear Regression Analysis of Correlation

To provide more information regarding susceptibilities of baseline clinical isolates to HCV nonnucleoside polymerase inhibitors, the susceptibilities of the baseline clinical isolates were compared pairwise among the three NNIs. Linear regression analysis was performed with the mean EC₅₀ values of each clinical isolate obtained in the transient phenotypic assays (Fig. 4). The correlations between the susceptibilities of the clinical isolates to any of the three NNIs tested were extremely poor as the coefficients of determination (r²) ranged between 0.0001 and 0.043. The P-values from the Pearson correlation of each analysis are much greater than the preset threshold value alpha of 0.05 (≥0.17). These data indicate there is no significant correlation among the susceptibilities to the HCV inhibitors tested in this study.

Figure 4. Correlation between different HCV inhibitors. The x-axis and y-axis represent mean EC₅₀ values from the corresponding NS5B chimeric clinical isolates. The line (–) represents the best unconstrained regression through the points. Sample size (n), the coefficient of determination (r²) and the P-value of each correlation plot are indicated.

Discussion

In this study, the sensitivity of up to 51 clinical baseline isolates from untreated HCV GT-1-infected patients to a panel of nonnucleoside HCV polymerase inhibitors was determined to investigate the effect of HCV genetic diversity on the inhibitor's antiviral potency. Some natural variation in susceptibility among these isolates was observed with all three different classes of HCV polymerase inhibitors tested (benzofuran, benzothiadiazine and thiophene carboxylic acid). The degree of the variation in EC₅₀ was slightly different among these three drug classes of nonnucleoside HCV polymerase inhibitors. The EC₅₀ values in the 95th percentile were 4.5-, 6.0- and 7.5-fold higher than the 5th percentile EC₅₀ values for the thiophene carboxylic acid, benzofuran and benzothiadiazine, respectively. Interestingly, no significant difference in sensitivity between GT-1a and GT-1b was observed for the tested benzofuran and thiophene carboxylic acid. In contrast, the benzothiadiazine was significantly more potent against GT-1b than GT-1a. The differential potency against GT-1a vs GT-1b for only the benzothiadiazine, and the high assay reproducibility for the reference standard suggests that the natural variation in drug susceptibility observed among baseline clinical isolates is not caused by assay variation. In addition, the results in the study are consistent with the findings of previous reports that described variable activity for HCV nonnucleoside polymerase inhibitors among clinical baseline isolates using similar phenotypic analysis assays.^[38,39]

Given the observation of the natural variation in drug susceptibility to these three classes of nonnucleoside inhibitors, it is possible that this natural variation may cause variable antiviral response among different patients. For example, one dose may be optimal for patients containing more sensitive variants, but this same dose may be suboptimal for patients who have less susceptible variants. Indeed, variable response to some of the HCV nonnucleoside polymerase inhibitors among different patients has been observed in clinical studies, especially in lower-dose groups. Furthermore, consistent with the in vitro findings of less potent activity against GT-1a than GT-1b for the benzothiadiazines in this study, the response in HCV GT-1a-infected patients was poorer than the GT-1b-infected patients during monotherapy with ABT-333 and ANA-598 (both benzothiadiazines).^[9,10] In addition to the susceptibility of baseline isolates, the pharmacokinetics of the drug also influences response. The natural variation in drug susceptibility coupled with pharmacokinetic information could be used to predict the response to drug treatment and provide guidance on the drug concentration needed for achieving a maximal response. If sufficiently high levels of drug exposure relative to the cluster of EC₅₀ values is achieved, the natural variation in drug susceptibility would be of less concern. Finally, the natural variation in baseline susceptibility could provide a threshold for defining abnormal reductions in drug susceptibility and serve as an indicator for an increased probability of drug resistance.

Sequence analysis of NS5B revealed C316N and S556G double mutants in 2 of the 14 GT-1b isolates. Phenotypic analysis of the one isolate with sufficient replication capacity to test drug susceptibility had the highest EC₅₀ against the benzofuran. This finding is consistent with previous reports, demonstrating that C316N confers a low-level of resistance to the benzofuran.^[26] However, the benzofuran susceptibility of this isolate was well within the natural variation of the GT-1a isolates and it had wild-type susceptibility to the benzothiadiazine tested in this study. Similarly, no significant change in EC₅₀ to the benzothiadiazine was seen in the isolate containing S556S/G. Previous studies showed that both C316N and S556G were associated with reduced susceptibility to other compounds of the benzothiadiazine class.^[23,24,26] The discrepancy may be due to the fact that the benzothiadiazine compound tested in this study has subtle differences in chemical structure with the benzothiadiazine compounds in previous studies resulting in a slightly different interaction with the NS5B polymerase.

In contrast to the above three GT-1b isolates, NS5B sequence analysis did not identify any of the mutations that are known to confer resistance to the tested compounds in 52 of 55 patient samples (41 GT-1a and 11 of 14 GT-1b). In addition, no clear pattern of the NS5B sequence was revealed in baseline clinical isolates with higher EC₅₀ values (data not shown). Thus, the natural variation in drug susceptibility observed in this study may be caused by the intrinsic genetic diversity of HCV.

The mean EC₅₀ values derived from each baseline clinical isolate against the benzofuran, benzothiadiazine and thiophene carboxylic acid were compared between drugs using the statistical regression analysis of correlation. Overall, the correlation was poor between any of the three compounds. These results suggest that the HCV variants which are less susceptible to one of these three classes of nonnucleoside polymerase inhibitors may not be less sensitive to the other two different classes of nonnucleoside polymerase inhibitors. Therefore, a combination of two or three different classes of nonnucleoside polymerase inhibitors may be advantageous for maximal antiviral response and reducing the selection of resistance. However, other aspects including synergistic/antagonistic inhibitory effects, drug–drug interactions and overlapping toxicity profiles should be taken into consideration for potential combination therapy strategies. The lack of correlation between two different classes of nonnucleotide polymerase inhibitors agrees with the fact that these three nonnucleoside inhibitors target different sites of the HCV polymerase and also exhibit different resistance profiles.

In summary, the activity of three classes of nonnucleoside polymerase inhibitors (benzofuran, benzothiadiazine and thiophene carboxylic acid) against a panel of baseline clinical isolates was evaluated using a replicon-based transient replication assay. Some variation in drug susceptibility was observed for all three classes of nonnucleoside polymerase inhibitors. However, there was no correlation between susceptibilities from one compound to another among those tested. Our findings suggest that the existing natural variation in baseline susceptibility should be taken into account for the optimal dose selection for the development of future nonnucleoside polymerase inhibitors. The lack of correlation between drug susceptibilities supports the combination of these different classes of HCV nonnucleotide polymerase inhibitors in the clinic.

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Cirrhosis Tied to Increased Risk of Liver Cancer in Diabetics

By David Douglas

NEW YORK (Reuters Health) Dec 26 - Cirrhosis and hepatitis are associated with the occurrence of hepatocellular carcinoma (HCC) in patients with diabetes, and hepatitis C is of particular importance, Taiwanese researchers report in a November 15 online paper in The American Journal of Gastroenterology.

As Dr. Shih-Wei Lai told Reuters Health by email, "In our study, diabetic patients comorbid with liver cirrhosis, hepatitis B, and hepatitis C were at significantly increased risk of developing hepatocellular carcinoma, and the risk associated with hepatitis C was stronger than that with hepatitis B."

Dr. Lai of China Medical University, Taichung and colleagues note that there is accumulating evidence that patients with diabetes mellitus are more prone to cancer in general and liver cancer in particular. An American study indicated that the risk of HCC in diabetic patients was more than twice that in non-diabetics, and there have been some similar findings in Taiwan.

To clarify the effect of diabetes on HCC risk, the team examined a health insurance database covering the years 2000 to 2005 and used the information to compare 19,349 newly diagnosed diabetes patients with 77,396 matched controls. Where possible, they were followed until the end of 2008.

The HCC incidence was doubled in diabetics compared with controls (21.0 versus 10.4 per 10,000 person-years), which translated to an adjusted hazard ratio of 1.73, the team found. Hazard ratios were also significantly and independently increased by being male (2.32), and having cirrhosis (8.65), hepatitis B (2.52), and hepatitis C (5.61).

Stratified analyses showed that subjects with diabetes and cirrhosis along with hepatitis C had the greatest elevation in risk (hazard ratio, 72.4).

The researchers then went on to examine the association between HCC and anti-diabetic medication. After adjustment, metformin use was associated with significant protection (hazard ratio, 0.49). This was also true of thiazolidinediones (hazard ratio, 0.56). Taking insulin, sulfonylureas, and other agents also reduced the risk, but not significantly.

Overall, given the influence of the studied comorbidities, concluded Dr. Lai, "These observations suggest that patients with these disorders may be the high-risk group that deserves to be closely monitored. The importance of hepatitis C should not be overlooked."

SOURCE: http://bit.ly/vHt1Dv

Am J Gastroenterol 2011.

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Hepatic steatosis in chronic hepatitis C patients infected with genotype 2 is associated with insulin resistance, hepatic fibrosis and affects cumulative positivity of serum hepatitis C virus RNA in peginterferon and ribavirin combination therapy

Authors: Itoh, Yoshito¹; Nishimura, Takeshi¹; Yamaguchi, Kanji¹; Yokomizo, Chihiro¹; Fujii, Hideki¹; Minami, Masahito¹; Nagao, Yasuyuki²; Sumida, Yoshio³; Hashimoto, Hiroaki⁴; Umemura, Atsushi⁴; Shima, Toshihide⁴; Okanoue, Takeshi⁴; Yoshikawa, Toshikazu¹

Source: Hepatology Research, Volume 41, Number 12, 1 December 2011 , pp. 1145-1152(8)

Publisher: Wiley-Blackwell

Abstract:

Aim: Hepatic steatosis is one of the factors limiting the virological response to interferon-based antiviral therapy for chronic hepatitis C (CH-C) patients infected with genotype 1, while contradictory results have been reported for genotype 2. We aimed to clarify the effect of hepatic steatosis on therapeutic outcome and cumulative positivity of serum HCV RNA in CH-C patients infected with genotype 2 treated by peginterferon (PEG-IFN)α2b and ribavirin (RBV) combination therapy.

Methods: A total of 74 treatment-naïve non-cirrhotic CH-C patients infected with genotype 2 who received PEG-IFNα2b and RBV according to the standard regimen were divided into hepatic steatosis 0-10% and >10% groups. The clinical backgrounds, sustained virological response (SVR) rates and cumulative positivity of serum HCV RNA were compared between the two groups.

Results: Among the 74 patients, 61 (82.4%) had hepatic steatosis 0-10% and 13 (17.6%) had hepatic steatosis >10%. Scores of homeostasis model assessment-insulin resistance and hepatic fibrosis were higher in patients with hepatic steatosis >10% than hepatic steatosis 0-10% (P = 0.040 and 0.042, respectively). Non-SVR was more frequent in patients with hepatic steatosis >10% than hepatic steatosis 0-10% (P = 0.003). Cumulative positivity of serum HCV RNA was significantly higher in patients with hepatic steatosis >10% than hepatic steatosis 0-10% (P = 0.004).

Conclusions: In CH-C patients infected with genotype 2 treated by PEG-IFNα2b and RBV combination therapy, hepatic steatosis >10% was associated with increased insulin resistance, advanced hepatic fibrosis and higher cumulative positivity of serum HCV RNA, which lead to a higher risk of non-SVR.

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1872-034X.2011.00886.x

Affiliations:1: Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kyoto2: Department of Gastroenterology, Matsushita Memorial Hospital3: Department of Gastroenterology and Hepatology Nara City Hospital, Nara, Japan4: Hepatology Center, Saiseikai Suita Hospital, Osaka

Publication date: 2011-12-01

Source

Favorable factors for re-treatment with pegylated interferon α2a plus ribavirin in patients with high viral loads of genotype 1 hepatitis C virus

Authors: Tamori, Akihiro¹; Kioka, Kiyohide²; Kurai, Osamu³; Sakaguchi, Hiroki⁴; Enomoto, Masaru¹; Fujii, Hideki¹; Kobayashi, Sawako¹; Iwai, Shuji¹; Morikawa, Hiroyasu¹; Yamaguchi, Seiko³; Kawasaki, Yasuko²; Oka, Hiroko³; Tanaka, Yasuhito⁵; Kawada, Norifumi¹

Source: Hepatology Research, Volume 41, Number 12, 1 December 2011 , pp. 1169-1177(9)

Publisher: Wiley-Blackwell

Abstract:

Aim: Effect of re-treatment for pegylated interferon (PEG-IFN) plus ribavirin was not fully evaluated. We examined the effects of re-treatment with PEG-IFN plus ribavirin in patients with high viral loads of genotype 1 hepatitis C virus who failed to achieve a sustained virological response (SVR) with combination therapy.

Methods: We examined 38 patients who were re-treated with PEG-IFN α2a plus ribavirin for more than 60 weeks, among whom 14 were non-responders and 24 were relapsers after previous treatment with PEG-IFN α2b plus ribavirin. IL28B genotyping was done in 21 patients.

Results: The overall SVR rate was 34%. Analysis of baseline characteristics showed that the relapsers had a significantly higher SVR rate than the non-responders (50.0%, 12/24 vs. 7.1%, 1/14, respectively, P = 0.012) The SVR rates of re-treated patients who had turned hepatitis C virus (HCV) RNA-negative at weeks 8, 12, 24, and 48 of the previous therapy were 67% (4/6), 67% (4/6), 29% (2/7), and 25% (1/4), respectively. Re-treatment achieved an SVR in five of 12 patients with IL28B major alleles and three of nine patients with IL28B minor alleles. During the re-treatment, patients with complete viral suppression at week-12 achieved a significantly higher SVR rate (P = 0.001).

Conclusions: Re-treatment with PEG-IFN α2a plus ribavirin therapy is effective in patients who relapse after a course of PEG-IFN α2b plus ribavirin therapy. Re-treatment is a particularly useful option for patients who achieve early viral clearance during previous therapy.

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1872-034X.2011.00887.x

Affiliations:1: Department of Hepatology, Osaka City University Graduate School of Medicine2: Department of Hepatology Osaka City General Hospital3: Department of Gastroenterology and Hepatology, Osaka City Juso Hospital, Osaka4: Internal Medicine, Izumi Municipal Hospital, Izumi5: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Publication date: 2011-12-01

Source

Prediction of sustained response to low-dose pegylated interferon alpha-2b plus ribavirin in patients with genotype 1b and high hepatitis C virus level using viral reduction within 2 weeks after therapy initiation

Authors: Tamai, Hideyuki; Shingaki, Naoki; Shiraki, Tatsuya; Tukuda, Hiroshi; Mori, Yoshiyuki; Moribata, Kosaku; Enomoto, Shotaro; Deguchi, Hisanobu; Ueda, Kazuki; Inoue, Izumi; Maekita, Takao; Iguchi, Mikitaka; Yanaoka, Kimihiko; Oka, Masashi; Ichinose, Masao

Source: Hepatology Research, Volume 41, Number 12, 1 December 2011 , pp. 1137-1144(8)

Publisher: Wiley-Blackwell

Abstract:

Aim: Continuation of pegylated interferon (PEG-IFN) plus ribavirin at the recommended dose is difficult in elderly patients and/or patients with cytopenia or complications. Whether the therapeutic efficacy of low-dose PEG-IFN plus ribavirin therapy could be predicted based on virological response within 2 weeks of therapy initiation was evaluated.

Methods: A total of 106 patients with a high viral load of genotype-1b hepatitis C virus (HCV) underwent low-dose PEG-IFN plus ribavirin therapy. PEG-IFN alpha 2b (0.75 µg/kg per week) and ribavirin (600-800 mg/day) were administered for 48 weeks.

Results: Sustained virological response (SVR) was achieved in 37%, and treatment was discontinued in 9%. On univariate analysis of SVR-contributing factors, significant differences were noted in the white blood cell count, platelet count, fibrosis markers, and viral reduction within 2 weeks from therapy initiation. On multivariate analysis, the platelet count and the reduction in the HCV core antigen level at week 2 were independent factors. The positive predictive value (PPV) and the negative predictive value (NPV) for SVR based on a 1-log or greater HCV-RNA level reduction at week 2 were 65% and 90%, respectively, and those based on HCV core antigen level at week 2 were 64% and 97%, respectively. PPV and NPV based on a 2-log or greater reduction of the RNA level were 86% and 67%, respectively, and those based on the core antigen level were 93% and 69%, respectively.

Conclusion: Evaluation of viral reduction at week 2 after therapy initiation is useful for predicting SVR to low-dose PEG-IFN plus ribavirin therapy.

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1872-034X.2011.00879.x

Affiliations:1: Second Department of Internal Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama city, Wakayama 641-0012, Japan

Publication date: 2011-12-01

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Editorial: staging liver fibrosis in hepatitis C: a challenge for this decade

Am J Gastroenterol. 2011 Dec;106(12):2121-2. doi: 10.1038/ajg.2011.343

Lai M, Afdhal NH.

Source

Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

Abstract

The limitations of and the invasive nature of liver biopsy has spurred extensive interest in the development of non-invasive tests to measure liver fibrosis in patients with chronic hepatitis C. Clinically applicable non-invasive tests, including radiological studies, elastography, and serum markers, all of which perform extremely well in excluding significant disease and diagnosing cirrhosis. FibroScan and acoustic radiation force impulse elastography are two elastography-based tests that show promise. In this new era of increased cure rates with newly Food and Drug Administration-approved drugs and the availability of multiple non-invasive tests of liver fibrosis, we anticipate a decreasing need for liver biopsies in the management of chronic hepatitis C.Am J Gastroenterol 2011; 106:2121-2122; doi:10.1038/ajg.2011.343.

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Hepatitis C Virus Infects the Endothelial Cells of the Blood-Brain Barrier

Gastroenterology. 2011 Nov 30. [Epub ahead of print]

Fletcher NF, Wilson GK, Murray J, Hu K, Lewis A, Reynolds GM, Stamataki Z, Meredith LW, Rowe IA, Luo G, Lopez-Ramirez MA, Baumert TF, Weksler B, Couraud PO, Kim KS, Romero IA, Jopling C, Morgello S, Balfe P, McKeating JA.

Source

Hepatitis C Research Group, Institute for Biomedical Research, University of Birmingham, Birmingham, UK.

Abstract

BACKGROUND & AIMS:

Hepatitis C Virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS.

METHODS:

We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells using quantitative PCR and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture-derived HCV were used to study the ability of endothelial cells to support viral entry and replication.

RESULTS:

Using quantitative PCR we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hCMEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor-BI, and Claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis.

CONCLUSIONS:

Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.

Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

Source

Drug Trials Not Representative, Researchers Charge

By Emily P. Walker, Washington Correspondent, MedPage Today
Published: December 27, 2011

Few major randomized, controlled clinical trials examine the effects of a drug in patients who have multiple chronic conditions, even though more than one-quarter of all Americans are living with at least two chronic health conditions, researchers reported.

The proportion is even greater for older individuals, two out of three of whom are likely to have at least two chronic health conditions, according to Alejandro Jadad, MD, and colleagues from the Centre for Health, Wellness and Cancer Survivorship at the University Health Network in Toronto.

That means that most trials on which the FDA bases its approval of new drugs are not generalizable to the U.S. population, they wrote in a research letter published in the Dec. 28 issue of the Journal of the American Medical Association.

Jadad and his colleagues examined all randomized controlled studies that dealt with an intervention for a long-lasting or chronic disease or condition that were published from January-March in 1995, 2000, 2005, and 2010 in five major peer-reviewed medical journals -- BMJ, CMAJ, JAMA, The Lancet, and the New England Journal of Medicine -- as well as six journals that focus on the most prevalent chronic conditions, including Circulation and Annals of General Psychiatry.

Only six of the 284 published trials analyzed (2.1%) explicitly included patients with multiple chronic conditions, and that percentage didn't change much from 1995 to 2010. In 179 of the randomized controlled trials, patients with multiple medical conditions were explicitly excluded from the trial.

Patients with multiple conditions were mentioned often in trial reports, although not actually included in the trial. About 70% of the published trial reports mentioned multiple coexisting diseases; with general medical journals describing them more often than specialized journals (72% versus 69%; P=0.02).

The letter authors concluded that few randomized controlled clinical trials published in the last 15 years have included patients with multiple chronic conditions.

Although the study was small, the authors said the finding "invites reflection about the risk of unintended harm from inappropriate generalization of trial results conducted in populations with a single disease."

"Given the possible drug-to-drug, drug-to-disease, and disease-to-disease interactions that remain unexamined, most of the evidence gathered to date by [randomized controlled clinical trials] is of limited value to guide decisions," they wrote.

The study authors said it may be useful for the FDA to have drug companies include subgroups of patients with the most common combinations of diseases in their drug development process; to observe safety outcomes of adding a new drug to patients who are already medicated for other conditions; and to have post-marketing studies that include the risk stratification to allow for meta-analyses across populations, such as those with multiple conditions.

The study was supported by funds from the Canada Research Chair in eHealth Innovation, the University of Toronto, and the University Health Network.

One of the letter writers, Alejandro Jadad, reported being a consultant to Foresight Links Corporation and that he receives royalties from Wiley for a book on randomized controlled trials.

None of others reported having any financial disclosures.

Primary source: Journal of the American Medical Association
Source reference:
Jahad A, et al "Consideration of multiple chronic diseases in randomized controlled trial" JAMA 2011; 306(24): 2670-2674.

Source

Researchers strike gold with nanoparticle virus test

Nehal Lasheen 27 December 2011 | EN

[CAIRO] An Egyptian research team has won a prize for developing a hepatitis C test using gold nanoparticles. The test could become a cheap and rapid way to screen people and blood banks for the virus.

Hassan Azzazy, chair of the chemistry department at The American University in Cairo (AUC), and his team, won third place at the Intel Global Challenge held at the University of California, Berkeley, in the United States, last month (7–10 November) winning US$10,000.

"Nanoparticles are promising tools for developing the next generation of diagnostic assays because of their unique properties," Azzazy told SciDev.Net. He said gold nanoparticles exhibit a phenomenon known as surface plasmon resonance, which causes them to change colour from an intense red to blue when they aggregate. The test works by adding a target agent that binds to viral genetic material, forcing the nanoparticles to aggregate and turning the solution blue.

Azzazy said that "conventional detection of active hepatitis C generally requires two tests that take from three to four days and cost about US$78 per sample, while the nanogold test can generate similar results in less than one hour and for one-tenth of the cost. One gram of gold tetrachloride costs US$260 and is enough for 10,000 samples."

According to the WHO, hepatitis C infects 200 million people worldwide, but is especially prevalent in the Middle East and North Africa (MENA) with about 9.2 million individuals infected. For most MENA countries, the prevalence is between one and two per cent; the exception is Egypt where estimated prevalence is 14 per cent for adults aged 15–60.

"Early identification and treatment of infected patients is critical to reducing transmission of the disease, and a fast and relatively cheap new assay will be of great help," said Hala Gabr, professor of haematology at Cairo University.

Amr Abul-Fotouh, lecturer in tropical medicine and hepatology at Cairo University, added: "Although the nanogold test is a good one, it is not quantitative so it cannot be used to treat and follow up patients, as this needs the quantity of the virus to be detected by a PCR [polymerase chain reaction] test".

He pointed out that the new assay could be used in blood banks to check for hepatitis C. "We have seen that about 20 per cent of Egyptian blood donors test positive for the virus," he said.

Sherif Shawky, a doctoral fellow at AUC, and a member of the research team, told SciDev.Net that the technology can be adapted for detecting other infectious agents such as tuberculosis, and for use as cancer biomarkers. A spin-off company, NanoDiagX, has been set up to develop these bio-nanotechnology platforms.

Source

Human Trials Initiated for New HIV Vaccine

Clade C HIV Envelope Protein Being Tested in Phase I Clinical Trial

SEATTLE, Dec. 21, 2011 /PRNewswire-USNewswire/ -- In the first clinical trial of an injectable vaccine containing trimeric HIV envelope protein (gp140) relevant to the predominant strain of HIV in Africa, researchers from four UK academic centers (St George's University London, Imperial College, Hull York Medical School (HYMS; University of York) and the Medical Research Council Clinical Trial Unit) and from the Infectious Disease Research Institute (IDRI) have come together to evaluate whether the vaccine is safe for use in human volunteers. If the vaccine does prove to be safe, and induces appropriate immunity, it could be considered for further testing and eventually be evaluated for its effectiveness as a vaccine for protecting women against HIV infection in Sub-Saharan Africa.

The trial, which is funded by the Wellcome Trust and goes by the name MUCOVAC2, is evaluating a vaccine that contains the HIV trimeric gp140 protein CN54, representative of Clade C strains of the virus. This clade of HIV is the most prevalent type of virus in Sub-Saharan Africa and responsible for the greatest number of infections globally. The trimeric protein represents the major target for antibodies on the viral surface.

The vaccine candidate will be formulated with an adjuvant known as GLA, developed by IDRI to enhance immune responsiveness following intramuscular injection. GLA formulations have been previously tested clinically with promising results.

Globally, Clade C HIV has caused the world's worst HIV epidemics and has infected half of the 34 million people living with HIV. In Sub-Saharan Africa, Clade C virus has infected the majority of adults with HIV and is predominant in India, China and South America. Vaccine candidates relevant to the Sub-Saharan epidemic are critically important to prevent large scale HIV infection in the fight against the global HIV epidemic.

MUCOVAC2, which is now screening potential participants, will enroll 36 healthy, HIV-negative women aged 18-45 years at St George's University of London and the HYMS Experimental Medicine Unit at York Hospital. Researchers will evaluate the vaccine's safety and determine the quality and magnitude of induced immune responses. The study is expected to take less than a year to complete with results available early 2013.

Women will be randomly assigned to receive the vaccine by intramuscular injection, intranasal immunization through the application of liquid drops to the nose, or a combination of intramuscular injection followed by intravaginal immunization through the application of a gel-based formulation. This will enable researchers to compare the safety and levels of induced antibodies in the blood and vaginal secretions generated by the different vaccine approaches.

Leading the study for St George's is Dr. Catherine Cosgrove, with Professor Charles Lacey leading the study at York.

"Globally, women comprise half of the 34 million people living with HIV. In Sub-Saharan Africa, women represent nearly 60 percent of adults with the virus. Our collaboration marks an important juncture for the field as we begin to assess which routes of immunization may provide the best responses to protect women," remarked Professor Robin Shattock, who is Chair in Mucosal Infection and Immunity at Imperial College, and who leads the consortium which developed the MUCOVAC2 trial.

Additional information about MUCOVAC2 and other UK vaccine studies is available at http://www.helpmakehistory.mrc.ac.uk.

About IDRI – Translating science into global health solutions

IDRI is a Seattle-based not-for-profit organization committed to applying innovative science to the research and development of products to prevent, detect, and treat infectious diseases of poverty. By integrating capabilities — including early stage drug discovery, preclinical testing, manufacturing, and clinical trials — IDRI strives to create an efficient pathway bringing scientific innovation from the laboratory to the people who need it most. www.idri.org

SOURCE Infectious Disease Research Institute (IDRI)

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AASLD: Fever Signals Responsiveness to Pegylated Interferon for Hepatitis C

Liz Highleyman | Content provided by hivandhepatitis.com

Published Tuesday, 27 December 2011

People who have an increase in body temperature soon after starting interferon-based therapy for chronic hepatitis C virus (HCV) infection are more likely to experience early virological response, according to a presentation at the American Association for the Study of Liver Disease (AASLD) Liver Meeting last month in San Francisco.

Rather than directly targeting HCV, interferon works by stimulating the body's immune response. This can lead to a variety of flu-like symptoms including fever, loss of appetite, and muscle aches. But it is not known how these side effects relate to antiviral activity.

Hwalih Hanand colleagues from the National Institutes of Health evaluated whether temperature changes after starting interferon reflect responsiveness to pegylated interferon, as assessed by viral kinetics, serum cytokine levels, and treatment response.
The study included 60 previously untreated chronic hepatitis C patients who started standard therapy using pegylated interferon plus ribavirin. Half were men, nearly 60% were white, about 20% each were black and Asian, and the mean age was 52 years. A majority (57%) had difficult-to-treat HCV genotype 1, one-third had genotypes 2 or 3, and 10% had genotypes 4, 5, or 6.

The researchers measured body temperature with an oral thermometer before the first injection of pegylated interferon and at 8, 16, and 24 hours thereafter, and determined the maximum temperature increase from baseline within the first 24 hours, or Tmax.

Serum HCV viral load was measured at baseline, at 6, 24, 48, and 72 hours, and then weekly for the first 4 weeks of treatment, and used to calculate the first and second phase slope of HCV decline. Levels of interferon gamma-inducible protein 10 (IP-10) were also assessed for a subset of patients at baseline and at 6 and 24 hours after treatment initiation.
Results

• The average maximum temperature increase after starting interferon was 1.1°C.
• The average peak temperature was 37.9°C, and 33% of patients experienced fevers above 38.0°C within 24 hours.
• Overall, 42% of participants experienced rapid virological response (RVR) at week 4, 83% showed early virological response (EVR) at week 12, and 38% achieved sustained virological response (SVR) at 24 weeks after the end of treatment.
• There was a strong and significant positive correlation between maximum temperature (Tmax) and the first phase of viral decline.
• However, Tmax did not correlate with the second phase of viral decline.
• The correlation between Tmax and first phase viral decline was similar for people with genotype 1 and those with genotypes 2 or 3, with no difference in average Tmax across viral genotypes.
• There was a significant positive correlation between Tmax and IP-10 induction at 6 and 24 hours.
• Tmax was significantly higher on average for participants with the favorable IL28B rs129790860 "CC" gene pattern -- a known predictor of treatment response -- compared to those with the unfavorable "CT" or "TT" patterns (1.43 vs 0.84°C, respectively).
• Tmax predicted RVR at week 4 and EVR at week 12, though the correlation was weak.

"Temperature increase after the initial injection of pegylated interferon is closely associated with interferon responsiveness, as reflected by the correlation with serum IP-10 increase, virological decline, and IL28B genotype," the researchers concluded. "The lack of difference between genotypes pinpoints its association with host-responsiveness factors."

As an easily measurable marker, they suggested, temperature "may be incorporated as a surrogate of more expensive and elaborate tests in future models of responsiveness to interferon-based treatment."

Investigator affiliation: Liver Diseases Branch, NIDDK, NIH, Bethesda, MD.

12/27/11

Reference

H Han, M Noureddin, YJ Park, et al. Changes in Oral Temperature After the Initial Injection of Peginterferon Alfa-2a in Patients with Chronic Hepatitis C Reflect Host-Interferon Responsiveness. 62nd Annual Meeting of the American Association for the Study of Liver Disease (AASLD 2011). San Francisco, November 4-8. 2011. Abstract 487.

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AASLD: Upping Ribavirin Dose Does Not Increase Interferon Effectiveness in HIV/HCV Coinfected Patients

Liz Highleyman | Content provided by hivandhepatitis.com

Published Tuesday, 22 November 2011

Starting hepatitis C treatment with a double dose of ribavirin plus erythropoietin to manage anemia did not lead to higher rates of sustained response to interferon-based therapy in HIV/HCV coinfected people, researchers reported at the American Association for the Study of Liver Diseases Liver Meeting (AASLD 2011) this month in San Francisco.

An estimated one-third of HIV positive people are coinfected with hepatitis C virus (HCV), and dual infection is associated with faster liver disease progression and poorer response to interferon-based therapy.

Vincent Soriano from Hospital Carlos III in Madrid and colleagues conducted a study to determine whether increasing the usual dose of ribavirin might improve virological response in this patient population.

Ribavirin promotes sustained virological response (SVR) because it reduces the risk of relapse after the end of treatment. Studies have shown that 1000-1200 mg/day weight-adjusted ribavirin works better than a fixed 800 mg/day dose, but the effect of even higher doses is not well understood. Ribavirin can cause hemolytic anemia (destruction of red blood cells), so higher-than-normal doses must be used with caution.

The PERICO study included 357 HIV/HCV coinfected participants who had not previously received interferon for hepatitis C. About three-quarters were men and the average age was 43 years. About 60% had HCV genotype 1, 19% had genotype 3, 17% had genotype 4, and only 6% had genotype 2. About half had advanced liver fibrosis/cirrhosis, 43% had the favorable IL28B CC gene pattern, and 72% had high baseline HCV RNA > 500,000 IU/mL. Most were on antiretroviral therapy with undetectable HIV viral load, and the mean CD4 T-cell count was about 550 cells/mm³.

All participants received the standard 180 mcg/week dose of pegyalted interferon alfa-2a (Pegasys). In addition, they were randomly assigned to receive either standard 1000-1200 mg/day weight-adjusted ribavirin for the full course of treatment, or else a 2000 mg/day induction dose for the first 4 weeks along with weekly subcutaneous injections of 50,000 IU erythropoietin (Procrit or Epogen), then dropping down to the standard ribavirin dose and discontinuing erythropoietin.

Patients with rapid virological response (RVR) at week 4 were treated for the standard duration of 24 weeks for genotypes 2 and 3 or 48 weeks for genotypes 1 and 4. People without RVR were treated for 48 or 72 weeks, respectively. Participants with inadequate response at weeks 12 or 24 stopped treatment early.

Results

• In an intent-to-treat analysis, 43% of participants in the high-dose ribavirin induction arm achieved SVR 24 weeks after the end of treatment, compared with 47% in the standard therapy arm, not a significant difference.
• In an on-treatment analysis, the corresponding rates were 53% vs 57%, again not a significant difference.
• Premature treatment discontinuation occurred with similar frequency in both arms -- 51% vs 53%, respectively -- mostly due to virological failure.
• Other response predictors were associated with higher sustained response rates, as expected:
  • HCV genotype: 82% for genotypes 2 and 3, 42% for genotype 1b, 34% for genotype 4, and 31% for genotype 1a;
  • IL28B gene pattern: 74% for CC vs 35% for CT or TT;
  • Treatment completion: 80% for completion vs 31% for early discontinuation.
• In a multivariate analysis, SVR was significantly associated with HCV genotypes 2 and 3, IL28B CC, and low baseline HCV RNA.
• RVR was the best predictor of SVR.
• Despite differing doses, ribavirin plasma trough levels (lowest between doses) at week 4 were the same in both arms, at 2.3 mcg/mL.

Based on these findings, the investigators concluded, "Induction therapy with high ribavirin dosing along with erythropoietin does not improve SVR rates in HIV/HCV coinfected patients."

To explain the unexpected similar ribavirin levels regardless of dose, they suggested that pre-emptive erythropoietin "might blunt the benefit of ribavirin overdosing by enhancing erythrocyte uptake of plasma ribavirin."

Investigator affiliations: Infectious Diseases, Hospital Carlos III, Madrid, Madrid, Spain; Hospital Clínico San Cecilio, Granada, Spain; Hospital San Pablo, Barcelona, Spain; Hospital Gregorio Marañón, Madrid, Spain; Hospital 12 de Octubre, Madrid, Spain; Hospital Clinico San Carlos, Madrid, Spain; Hospital Virgen de La Victoria, Malaga, Spain; H Virgen Macarena, Sevilla, Spain; Hospital Universitario de Valme, Sevilla, Spain; Hospital Txagorritxu, Vitoria, Spain; Hospital Gral de Jerez, Jerez, Spain; Hospital do Meixoeiro, Vigo, Spain; Hospital Xeral-Cies, Vigo, Spain; Hospital Cruces, Bilbao, Spain; Hospital La Princesa, Madrid, Spain; Hospital Central de Asturias, Oviedo, Spain.

11/22/11

Reference

P Labarga, M Téllez, P Barreiro, V Soriano, et al. The Perico Trial: A Multicenter Randomized Controled Trial Comparing High Ribavirin (RBV) Induction vs Standard RBV Dosing in the Treatment of Chronic Hepatitis C in HIV-Coinfected Patients. 62nd Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2011). San Francisco, November 4-8. 2011. Abstract 247.

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Proteonomix, Inc. (PROT) Announces Completion of Payment for Its Clinical Trial of UMK-121 in Patients With End Stage Liver Disease

Dec. 27, 2011, 2:35 p.m. EST

MOUNTAINSIDE, NJ, Dec 27, 2011 (MARKETWIRE via COMTEX) -- PROTEONOMIX, INC. a biotechnology company focused on developing therapeutics based upon the use of human cells and their derivatives, announced today that it has completed all payments required from Proteonomix to commence a Clinical Study entitled "UMK-121 in Patients with Liver Disease."

As previously announced, the Company entered into an Agreement to conduct the clinical trial with the University of Miami. That Agreement required the University to pay expenses associated with the clinical study and The Company was required to assist financially with the clinical study.

Michael Cohen, President of the Company, stated: "The financing that was required to complete the Company's obligation with respect to the Trial was provided Friday, December 23, 2011. We previously thanked the University for its generous assistance in the agreement to conduct a clinical trial of UMK-121. The Company has previously described the terms of the agreement to license and develop and the patent application of the UMK-121 technology. The Company will work together with the University and the principal investigators to initiate the clinical study."

Mr. Cohen continued, "Our UMK-121 pharmaceutical therapy is advancing toward its first human clinical trial in ESLD ('End Stage Liver Disease') patients. We hope to provide patients who are suffering from this debilitating and mortal condition with alternatives. We believe that the commercialization of this technology can provide Proteonomix with significant revenue potential. According to United Network for Organ Sharing ('UNOS') there are over 100,000 patients on the transplant waiting list at any given time. According to the ('NIH') there are over 500,000 patients in end stage Kidney disease. According to Organ Donation and Transplantation ('NWHIC') over 60,000 Americans suffer from End Stage Liver Disease ('ESLD'). The Company will continue to provide information about the clinical study in the upcoming weeks and months consistent with our understanding with the University and ethical considerations."

About Proteonomix, Inc.:

Proteonomix is a biotechnology company focused on developing therapeutics based upon the use of human cells and their derivatives. The Proteonomix Family of companies includes Proteoderm, StromaCel, PRTMI and THOR Biopharma. Proteoderm, Inc. is a wholly owned subsidiary that has developed an anti-aging line of skin care products. StromaCel, Inc. develops therapeutic modalities for the treatment of Cardiovascular Disease (CVD). Proteonomix Regenerative Translational Medicine Institute, Inc. ("PRTMI") intends to focus on the translation of promising research in stem cell biology and cellular therapy to clinical applications of regenerative medicine. Proteonomix intends to create and dedicate a subsidiary to each of its technologies. Please also visit http://www.proteonomix.com/ , http://www.proteoderm.com/ , http://www.otcqb.com/ and http://www.sec.gov/ .

Forward-looking statements:

Certain statements contained herein are "forward-looking statements" (as defined in the Private Securities Litigation Reform Act of 1995). Proteonomix, Inc. cautions that statements made in this press release constitute forward-looking statements and makes no guarantee of future performance. Actual results or developments may differ materially from projections. Forward-looking statements are based on estimates and opinions of management at the time statements are made

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